Apoptosis marker antibodies and methods of use

ABSTRACT

Disclosed are antibodies that specifically recognize the new amino terminus of a protein cleaved by a protease during apoptosis. Methods of using and making the antibodies are also provided. The antibodies are particularly useful in methods of detecting apoptosis and testing candidate compounds for enhancing or inhibiting apoptosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/101,920, filed Sep. 24, 1998.

FIELD OF THE INVENTION

The present invention relates generally to the field of detecting andquantifying apoptosis. More particularly, the present invention relatesto antibodies to the newly formed amino terminus resulting from cleavageof proteins during the process of apoptosis and to the use of suchantibodies in detecting apoptosis in cells undergoing apoptosis or incells that have undergone apoptosis.

BACKGROUND OF THE INVENTION

Most eukaryotic cells have the ability to self-destruct by activation ofan intrinsic cellular suicide program referred to as programmed celldeath or apoptosis. The process of apoptosis involves a cascade ofcytoplasmic and nuclear events that result in a series of morphologicalchanges, and eventually cause the demise of the cell. Apoptosis ischaracterized by distinct biochemical and morphological changesexhibited by cells undergoing programmed cell death, including DNAfragmentation, plasma membrane blebbing, and cell volume shrinkage. Atthe molecular level, activation of one or more aspartate-specific,cysteine proteases (caspases) is proposed to be the critical signalrequired to carry out apoptotic cell death (Yang et al., AmericanJournal of Pathology, 152(2):379-389, 1998).

The caspases, also known as ICE (IL-1 β-converting enzyme)-likeproteases, can be divided into three subclasses: ICE/CED3 family,CPP32/Yama family and the Ich/Nedd2 family (Duan et al., J. Biol. Chem.,271:1621-1625, 1996). All family members share a high level of aminoacid sequence homology with ICE, and contain a conserved QACRGpentapeptide in which the cysteine participates in catalysis (Nicholson,Nature Biotech., 14:297-301, 1996). Furthermore, all of these proteasesare reported to require an aspartic acid residue at the substrate P1position (Jänicke et al., The EMBO J., 15(24):6969-6978, 1996).

CPP32 (Caspase 3) has been identified as one of the proteases thatcleaves poly(ADP-ribose) polymerase (PARP) (Schlegel et al., J. Biol.Chem., 271:1841-1844, 1996; Nicholson et al., Nature, 376:37-42, 1995).PARP is one of the enzymes associated with DNA repair. Cleavage of theapproximately 116 kilodaltons (“kd”) PARP protein into fragments ofabout 89 kd and about 27 kd has been reported to contribute to the DNAfragmentation that is characteristic of apoptosis (Kayalar et al., Proc.Natl. Acad. Sci. USA, 93:2234-2238, 1996). Therefore, the identificationof the about 89 kd or the about 27 kd fragments resulting from thecleavage of PARP within a cell is an indication that the cell isundergoing or has undergone apoptosis.

Proteins consist of macromolecules of amino acids linked by peptidebonds, to form polypeptide chains. Each amino acid in the chain consistsof a carbon atom to which are attached four different groups (—R, —H,—NH₂, and —CO₂H), wherein the identity of R varies from one amino acidto another. The peptide bond links each amino acid to the next aminoacid in the chain through a covalent bond formed between the —CO₂H groupof one amino acid and the —NH₂ group of the next amino acid, with H₂O abyproduct of the reaction. Every polypeptide chain has two terminalamino acid residues, one at each end of the chain. The end of the chainwith a —CO₂H group which has not been linked to another amino acid isreferred to as the “carboxy terminus” or “C-terminus”. The end of thechain with a —NH₂ group which has not been linked to another amino acidis referred to as an “amino terminus” or “N-terminus”. When an enzymesuch as caspase cleaves PARP, it breaks a peptide bond in a polypeptidechain of the protein, creating one fragment with a new carboxy terminusand another fragment with a new amino terminus.

One reference, WO 98/21590, describes methods of detecting apoptosis byusing antibodies that bind to the amino acids at the newly createdcarboxy termini of polypeptides generated by the cleavage of proteins bythe caspase family of proteases. Because caspases cleave immediatelycarboxy-terminal of a characteristic four amino acid recognition site,the newly created carboxy terminus of a caspase cleaved protein consistsof the last amino acid of the recognition site. WO 98/21590 describesthe production of antibodies following immunization of rabbits with apolypeptide comprising a caspase recognition site (GDEVD) at its carboxyterminus. Although the antibodies of WO 98/21590 appear to recognize acleaved fragment of PARP, these antibodies were cross-reactive withother proteins as well. This cross-reactivity is likely to result ininaccurate determinations of apoptosis in cells or cellular lysates.

In another attempt to produce antibodies that are specific to apoptoticfragments of PARP, Sallmann et al., Biochem. Cell Biol., 75:451-456,(1997) immunized rabbits with synthetic polypeptides corresponding tothe newly created carboxy terminus and amino terminus of PARP that areformed following cleavage of PARP by a caspase. Although the polyclonalantibodies produced by Sallmann et al. were able to distinguish betweenthe two (carboxy terminal and amino terminal) apoptotic fragments ofPARP, the antibodies were not able to distinguish between the cleavedfragment and uncleaved PARP. Therefore, the antibodies produced bySallmann et al. are not specific to epitopes produced in apoptotic cellsbecause they are immunoreactive with the uncleaved PARP present innon-apoptotic cells.

Therefore, there is a need for antibodies that are specifically able todistinguish apoptotic events in cells. These antibodies will enable moreaccurate results in methods for detecting apoptosis. Because apoptosis,or the inability of cells to undergo apoptosis, is associated with anumber of disorders and diseases including cancer, neurodegeneration,autoimmunity, heart disease and others (reviewed in Hetts, JAMA,279(4):300-307, 1998), improved methods of detecting apoptosis willprovide a better understanding of these diseases and will be useful inscreening potential therapeutic agents that may induce or preventapoptosis.

BRIEF SUMMARY OF THE INVENTION

The present invention seeks to overcome limitations in the prior art byproviding methods and compositions related to antibodies that recognizespecific epitopes that are indicators of apoptosis. Antibodies wereproduced that are specific to a new epitope (neoepitope) formed inapoptotic cells. The neoepitope is a product of cleavage of a protein ata specific cleavage site by a protease during apoptosis.

The present invention involves the use of antibodies raised against aneoepitope which comprises a polypeptide sequence which is homologous toonly one end of one of the fragments of a protein cleaved duringapoptosis. Every protein consists of a chain of amino acids. Each aminoacid covalently linked to the next amino acid in the chain through apeptide bond. A peptide bond links the amine residue of one amino acidwith the carboxyl residue of the next amino acid in the chain. Theresulting chain has an unattached amine residue at one end, and anunattached carboxy residue at the other end. When the protein iscleaved, and the chain of amino acids broken at that point, twofragments of the protein are formed, one with a new carboxy terminus,and the other with a new terminal amine. In the present invention, theneoepitope comprises the new amino terminus at the beginning of thecarboxy terminal fragment.

By immunizing animals with a polypeptide comprising a sequence of theamino acids of the newly formed amino terminus of a fragment of aprotein formed by cleavage of the protein during apoptosis, antibodieswere isolated that recognized the cleaved form of the protein but failedto recognize the uncleaved form. This result is surprising becauseattempts by others to produce specific antibodies to a new aminoterminus of PARP formed after cleavage by a caspase failed because theantibodies were still immunoreactive with the uncleaved protein(Sallmann et al., Biochem. Cell Ciol., 75:451-456, 1997). Furthermore,the isolated antibodies of the present invention show particular utilityin methods of detecting apoptosis.

The present invention includes compositions comprising an isolatedantibody that is immunoreactive with a neoepitope produced in a cellundergoing apoptosis. As used herein, the term immunoreactive means thatthe antibody is capable of binding the antigen with an affinity that isindicative of an immune reaction to the antigen. Such affinities arewell known to those of skill in the art and include affinities of 10⁵ to10¹⁴ M^(−1.) Methods of determining the affinity of an antibodycomposition are described in Day, Advanced Immunochemistry, (2^(nd)edition) Wiley-Liss, New York, N.Y. (1990).

Although binding the neoepitope is an important aspect of the presentinvention, it is also important that the antibody fails to bind theuncleaved protein. This ability to distinguish between the cleaved anduncleaved form of a protein gives the antibody its novel specificity todetect apoptotic cells, as the neoepitope is at a detectable level inapoptotic cells but below a detectable level in non-apoptotic cells.

The antibody may be a polyclonal or monoclonal antibody and recognizesthe new amino terminus of poly(ADP-ribose) polymerase (PARP) formed bycleavage of the protein by a caspase. A polypeptide comprising the aminoacid sequence of FIG. 1 (SEQ ID NO:2), when injected into a rabbit, wasable to cause production of antibodies that bound specifically to thecleaved form of PARP while failing to bind the uncleaved form.

Although the methods of the present invention may be employed with anumber of different proteases, the inventor has found that targets ofthe caspase family of proteases are particularly useful. The caspasefamily of proteases include ICE (caspase 1), caspase 3, caspase 7, andcaspase 8, and are reviewed in Nicholson, et al., TIBS, 22(8):299-306(1997) and Villa et al., TIBS, 22:388-393 (1997). In preferredembodiments, the neoepitope is the new amino terminus of a proteincleaved by caspase 7 or caspase 3.

An antibody of the present invention may be immunoreactive with anepitope in apoptotic cells from a number of species including chicken,bovine, murine, feline, canine, rat, equine, opine, and primate speciesincluding human. In preferred embodiments, the antibody isimmunoreactive with an epitope in apoptotic cells of a mammal,preferably human.

The apoptotic cell may be a cell of essentially any type known to becapable of the process of apoptotis including heart, lung, skeletalmuscle, neuronal, liver, kidney, pancreas, epithelial, or blood cell. Inpreferred embodiments, the blood cell is a leukemia cell such as HL-60.

The antibodies of the present invention may be used in methods ofdetecting apoptosis in a cell or group of cells. The methods disclosedherein allow the determination of apoptosis in a biological samplecomprising an individual cell or a group of cells. As used herein agroup of cells may be any collection of more than one cell such as ablood sample, tissue sample, biopsy, or tissue culture. A biologicalsample to be screened can be a biological fluid such as extracellular orintracellular fluid or a cell or tissue extract or homogenate. Abiological sample can also be an isolated cell (e.g., in culture) or acollection of cells such as in a tissue sample or histology sample. Atissue sample can be suspended in a liquid medium or fixed onto a solidsupport such as a microscope slide.

In some embodiments, the methods of detecting apoptosis in a cell orgroup of cells comprise obtaining a protein sample from the cell orgroup of cells, contacting the protein sample with an antibody which isimmunoreactive with a neoepitope in apoptotic cells, and screening thecell or group of cells to detect any of the antibody bound to theneoepitope. Detecting the antibody bound to the neoepitope in the sampleis indicative of apoptosis in the cell or group of cells, whereasfailing to detect the antibody bound to the neoepitope is indicative oflack of apoptosis in a cell or group of cells. Such methods includeimmunoassays such as Western blots, Enzyme Linked Immunosorbent Assay(“ELISA”), cell-based ELISA, filter-binding ELISA, inhibition ELISA,sandwich ELISA, immunostaining, immunoprecipitations, slot or dot blots,radioimmunoassays, scintillation proximity assays, Ouchterlony analysis,and fluorescent immunoassays. Some of the above methods require thecells to be lysed or processed to isolate proteins therefrom prior tothe detection step.

Other methods of the present invention for detecting apoptosis in cellsdo not require the step of acquiring a protein sample or lysate, butrather detect apoptosis in the cells or tissues themselves. Such methodsinclude immunohistochemistry, immunocytochemistry, and flow cytometry.

In some embodiments, the antibody of the present invention furthercomprises a label. A label is a molecule or substance that is attachedto the antibody that facilitates detection of the presence of theantibody. Labels are well known to those of skill in the art andinclude, but are not limited to, haptens such as biotin ornitro-iodo-phenyl, radioactive isotopes such as ¹²⁵I, ³H, ¹⁴C, ³²P, or³⁵S, enzymes such as alkaline phosphatase, horseradish peroxidase,β-galactosidase, or luciferase, and fluorescent or luminescentmolecules, such as fluorescein, rhodamine, phycoerythrin, Texas red,green flourescent protein or derivatives thereof.

Antibodies of the present invention comprising labels are particularlyuseful in methods of detecting apoptosis in a cell, group of cells, cellsample, or sample of a group of cells. In other embodiments, thepresence of an antibody of the present invention is detected by the useof a secondary antibody comprising a label.

Another embodiment of the present invention is a method of producing anantibody immunoreactive with a neoepitope produced in a cell undergoingapoptosis. This method comprises the steps of: (a) obtaining apolypeptide comprising an amino acid sequence corresponding with theamino terminus produced by cleavage of a protein by a protease duringapoptosis; and (b) administering the polypeptide to an animal underconditions suitable to provoke an immune response thereby producingantibodies to the polypeptide. In preferred embodiments of this lastmethod, antibodies obtained by such a method are removed from theanimal, or in the case of a chicken are removed from the egg, and testedto ensure that they are not immunoreactive with the uncleaved protein.

The methods of the present invention may be used for diagnosing adisease, disorder, or condition associated with cell apoptosis. Suchmethods comprise contacting a cell, tissue, group of cells, or samplesthereof, with an antibody of the present invention and detecting, orfailing to detect, the antibody bound to the neoepitope. Detecting theantibody bound to the neoepitope is indicative of apoptosis, whereasfailing to detect the antibody bound to the neoepitope suggestsapoptosis is not present in the cell, tissue, group of cells, or samplesthereof.

In yet another embodiment, the present invention provides methods ofscreening compounds to identify inhibitors of apoptosis. These methodscomprise exposing a sample of cells to conditions known to activateapoptosis in the cells, contacting the sample with a test or candidatecompound, contacting the sample with an antibody of the presentinvention, and quantifying or detecting the level of antibody bound tothe neoepitope in the sample. In preferred embodiments, a second sampleis induced into apoptosis and is subjected to the same steps as thefirst sample except the second sample is not contacted with the testcompound. The use of the untreated (by the test compound) second sampleallows one to compare the level of antibody bound to the neoepitope incells treated with the test compound, versus antibody levels in cellsnot treated with the test compound. The test compound is said to inhibitapoptosis if the level of the antibody in the first sample is less thanthe level of the antibody in the second sample.

In another embodiment, the present invention provides methods forscreening compounds to identify stimulators or inducers of apoptosis.Such methods comprise contacting a sample of cells with a test compound,contacting the sample with an antibody of the present invention, andquantifying or detecting the level of antibody bound to the neoepitopein the sample. In preferred embodiments, a second sample is subjected tothe same steps as the first sample except the second sample is notcontacted with the test compound. The use of the untreated second sampleallows one to compare the level of antibody bound to the neoepitope incells treated with the test compound versus cells not treated with thetest compound. The test compound induces apoptosis if the level of theantibody in the first sample is greater than the level of the antibodyin the second sample.

Also provided by the present invention are kits for detecting apoptosis.Specifically, the kits are for detecting apoptosis-generated proteinfragments in a sample. Such kits comprise an antibody of the presentinvention. In some embodiments, the antibody of the present inventioncomprises a label. In other embodiments, the antibody of the presentinvention is not labeled, but the kit further comprises a labeledsecondary antibody that is immunoreactive with the antibody of thepresent invention.

Other reagents that the kits of the present invention optionallycomprise antibodies immunoreactive with surface antigens (such as CD4,CD8, TCR, B220, Fas), antibodies immunoreactive with a proliferation orother marker antigens (such as p21, p53, Rb, PCNA, Ki-67, etc.), orreagents for the TUNEL (TdT-mediated dUTP Nick-End Labeling) reaction(Promega Corporation, Cat. No. #G3250, G7360).

Also provided, are methods of producing an antibody immunoreactive witha neoepitope comprising the amino terminus produced by cleavage of aprotein by a protease during apoptosis, but not immunoreactive with theuncleaved protein. Such methods comprise obtaining a polypeptidecomprising the amino terminus produced by cleavage of a protein by aprotease during apoptosis, administering the polypeptide to an animal toelicit antibody production against the polypeptide, and collecting theantibodies from the animal by methods known to those of skill in theart.

As used herein, “a” and “an” are defined to mean one or more.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of a polypeptide, identified hereinas SEQ ID NO:2, used to elicit antibodies specific to the neoepitope ofPARP, as illustrated in Example 1.

FIG. 2 identified as SEQ ID NO:5 shows the amino acid sequence fromamino acid position 196 to amino acid position 244 of the human polyADP-ribose polymerase protein (GenPept Acc. No. P09874). The caspaserecognition site (DEVD) is shown in bold type, followed by a verticalline showing the site where caspase cleaves the protein. The first sevenamino acids of the newly created amino terminus of the resulting about85 kd fragment produced from the cleavage of PARP with caspase areunderlined to show the sequence of the 7-mer polypeptide (SEQ ID NO:2)used in the production of antibodies specific to the terminus of thefragment, as illustrated in Examples 1 and 2.

FIG. 3 presents, in diagrammatic form, a structure of the polypeptideand linker molecule used in Example 1 to produce antibodies of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising antibodies thatare immunoreactive with (i.e., specific to) neoepitopes produced inapoptotic cells. Because an integral step of apoptosis is specificcleavage of specific cellular proteins by caspases, the new aminoterminus that results from such cleavage provides an excellent antigenfor the production of apoptosis-specific antibodies. Antibodies of thepresent invention, raised to a new amino terminus which results fromcleavage specific to apoptosis, such as antibodies raised to the newamino terminus of the about 85 kd fragment resulting from cleavage ofPARP after cleavage with caspase, are immunoreactive with the new aminoterminus produced by cleavage of the protein by a caspase duringapoptosis. Importantly, the antibodies are not immunoreactive with thenon-cleaved protein. Embodiments of the present invention include theantibodies themselves and antibodies with equivalent function, methodsof making the antibodies, methods of using the antibodies, and kits thatcontain the antibodies.

A. Caspases and Proteolytic Substrates

1. Proteases and Proteolytic Substrates During Apoptosis

Poly-ADP-ribose polymerase (PARP) is one of a number of proteins whoseproteolytic degradation is stimulated in cells undergoing apoptosis.Other such proteins include Keratin 18 (K18) (Caulin et al., J. Cell.Biol., 138(6):1379-94, 1997), MEKK-1 (Cardone et al., Cell,90(2):315-23, 1997), DNA replication complex C, DNA-dependent proteinkinase, protein kinase of presenilin 1 and 2, and spectrin (fodrin).These proteins are cleaved during apoptosis by a family of proteasesknown as caspases. A review of caspases and their protein targets isprovided by Nicholson and Thomberry, TIBS, 22(8):299-306 (1997).

The caspases recognize and cleave immediately after a distinct sequenceof amino acids within a protein. Based on knowledge of the recognitionsite of caspase 3 (CPP32), the cleavage site and subsequent new aminoterminus of the protein fragment corresponding to the carboxyl portionof PARP is predicted. A similar method may be used to predict the newamino terminus of other proteins cleaved by the caspases as well. A listof such proteins is shown in Table 1. It is contemplated that, given themethods disclosed herein, antibodies specific to the newly formed aminoterminus formed by caspase cleavage may be produced for any of theseproteins. Such antibodies are likely to be apoptosis-specificantibodies, which are readily determined by methods disclosed herein.

As provided herein, one would locate the caspase cleavage site withinthe amino acid sequence of the protein, produce a polypeptide thatcomprises the new amino terminus formed by cleavage of a caspase,immunize an animal with the polypeptide, collect the antibodies from theanimal, and screen the antibodies for the ability to recognize thecleaved form of the protein and not the uncleaved form of the protein.When the animal is a bird, such as a chicken, the antibodies arepreferably collected from a bird egg. Of course, one may chose not toscreen the antibodies, but it is preferred that one screens theantibodies to insure that they are specific to the new amino terminus.

Embodiments of the present invention include antibodies to the new aminoterminus of PARP produced by cleavage of PARP by a caspase duringapoptosis. Because this amino terminus is not present in uncleaved PARP,the antibodies are specific to a PARP fragment produced duringapoptosis. To produce the antibodies, a polypeptide comprising the newamino terminus of PARP are administered to animals in a mannercompatible with eliciting an immune response and production ofantibodies within the animals. It is contemplated that the antibodycould also be produced by injecting chickens and recovering antibodiesfrom the eggs. In a preferred embodiment, the polypeptide is GVDEVAK(SEQ ID NO:2, illustrated in FIG. 1). In a more preferred embodiment,this polypeptide further comprises the chemical structure illustrated inFIG. 3. It is preferred that the compound of FIG. 3 is conjugated to acarrier protein to increase its immunogenicity.

TABLE 1 Proteolytic substrates for caspases during apoptosis Site^(a)Cleaved protein Reference DEVD G PARP Kaufmann, S. H. et al.(1993)Lazebnik, et al.(1994) DEVD N DNA-PKcs Casciola-Rosen, L. A. etal.(1995,1996) Teraoka, H. et al.(1996) Le Romancer, M. et al.(1996)Song, Q. et al.(1996) DGPD G U1-70K snRNP Casciola-Rosen, L. A. etal.(1994) DXXD X HnRNP-C Waterhouse, N. et al.(1996) DEPD S SREBP (+)Wang, X. et al.(1995,1996) DELD S D4-GDI Na, S. et al.(1996) DXXD XHuntingtin Goldberg, Y. P. et al.(1996) DETD S DFF-45 site I(+) Liu, X.et al.(1997) DAVD T DFF-45 site II(+) Liu, X. et al.(1997) DEVD G DNA-RCC140 Ubeda, M. and Habener et al.(1997) Song, Q. et al.(1997) DMQD N PKCδ (+) Emoto, Y. et al.(1995) Gayhur, T. et al.(1996) DSID S Rb An, B.and Dou, Q.(1996) Jänicke, R. U. et al.(1996) Chen, W-D. et al.(1997)Tan, X. et al.(1997) DVPD C HDM2/MDM2 Chen, L. et al.(1996) DQTD S FAKCrouch, D. H. et al.(1996) DSLD L NuMA Hsu, H-L. et al.(1996) Casiano,C. A. et al.(1996) XXXD X Pro-caspase (+) DMQD N α-Fodrin Martin, S. etal.(1995) Vanags, D. M. et al.(1996) Cryns, V. L. et al.(1996) ELPD GActin Mashima, T. et al.(1995,1997) Chen, Z. et al.(1996) Song, Q. etal.(1997) Brown, S. B., et al.(1997) SRVD G Gas2 Broncolini, C., etal.(1995) VED N Lamins Lazebnik, Y. A. et al.(1995) P₄ P₁ Orth, K. etal.(1996) Takahashi, A. et al.(1996) Rao, L. et al.(1996) ^(a)Cleavagesites, indicated with the space, have been determined by proteinsequencing or mutational analysis (except for NuMA and α-fodrin, whichare sites corresponding to the molecular mass of cleavage products).(Nicholson and Thornberry, TIBS, 22(8):299-306, 1997)

The first thirty amino acids of the new amino terminus of thepolypeptide formed by the cleavage of human PARP during apoptosis isidentified herein as SEQ ID NO:1. The antibody of the present inventionis preferably raised to a polypeptide with a sequence of amino acidsbeginning with at least three of the first amino acids at the aminoterminus of SEQ ID NO:1, including but not limited to a 6-merpolypeptide identified by SEQ ID NO:3 and a 9-mer polypeptide identifiedby SEQ ID NO:4. The polypeptide of FIG. 1, consisting of an amino acidsequence identified by the first 7 amino acids, i.e. SEQ ID NO:2, fromthe new amino terminus of PARP in humans is preferred for use in theproduction of antibodies of the present invention. However, otherpolypeptides comprising the new amino terminus of PARP are suitable. Twocriteria should be considered when choosing polypeptides for theproduction of antibodies to the new amino terminus of PARP: (1) theamino terminus of the polypeptide should resemble that of the new aminoterminus of the about 89 kd of PARP formed by cleavage during apoptosisas closely as possible (i.e., should be glycine or structures chemicallyresembling glycine) and (2) the longer the polypeptide, the more likelyit will assume a structure similar to uncleaved PARP. Given the abovefactors, it is preferred that the polypeptide used in the presentinvention comprise 3 to 25 amino acids, more preferably 5 to 15 aminoacids, and even more preferably about 5 to about 10 amino acids and mostpreferably 7.

Because the antibodies raised to the new amino terminus of human PARPare able to recognize the new amino terminus of PARP of non-humanspecies, antibodies raised to polypeptides comprising the new aminoterminus of the PARP of other species may be cross-reactive with humanPARP. The caspase cleavage site of PARP of several species is shown inTable 2.

TABLE 2 Species (GenPept Acc. No.) Site New Amino Terminus Human(P09874) D E V D G V D E V A K Rat (P27008) D E V D G I D E V A K Mouse(P11103) D E V D G T D E V A K Bovine (P18493) D E V D G I D E V T KChicken (P26446) E E V D G N V V A T K Xenopus (P31669) D E V D G H S AA G K

The antibodies of the present invention may e used in methods ofdetecting and measuring apoptotic protelysis, in methods of identifyingand quantifying cells undergoing apoptosis in vitro and in vivo, andmethods of identifying compounds that stimulate or block apoptosis.

The methods of the invention may be used to diagnose a disease,disorder, or condition which is of either pathological ornon-pathological origin including chronic neurodegenerative diseases(such as Alzheimer's disease), cancer, sepsis, trauma, hypoxia, anoxia,ischemia (such as ischaemic reperfiusion injury), spinal trauma, headtrauma, lesion, rheumatoid arthritis, viral infections (such as EBV andHIV) and exposure to toxins.

In another embodiment, the present invention provides methods ofidentifying compounds that are useful in inhibiting or inducingapoptosis. Such methods may be in vitro, in vivo, or ex vivo.

In a preferred embodiment, the present invention provides immunoassaysfor detecting the result of proteolytic activity associated withapoptosis. Such immunoassays include, but are not limited to, Westemblots, ELISA, cell-based ELISA, filter-binding ELISA, inhibition ELISA,sandwich ELISA, immunostaining, immunoprecipitations, slot or dot blots,radioimmunoassays, scintillation proximity assays, Ouchterlony analysis,and fluorescent immunoassays (including flow cytometry) using antibodiesconjugates or antigen conjugates of fluorescent substances such asfluorescein, rhodamine, or phycoerythrin.

2. Methods for Preparing Antibody Compositions

The antibody compositions of the present invention may be produced by anumber of methods. Means for preparing and characterizing antibodies arewell known in the art (See e.g., Harlow, E. and Lane, D., Antibodies: ALaborato Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1988). Reference to antibodies throughout the specificationincludes whole polyclonal and monoclonal antibodies (“mAbs”), and partsthereof, either alone or conjugated with other moieties. Antibody partsinclude Fab and F(ab)₂ fragmented and single chain antibodies. Theantibodies may be made in vivo in suitable laboratory animals or invitro using recombinant DNA techniques (e.g., U.S. Pat. Nos. 4,975,369and 5,225,539).

Briefly, a polyclonal antibody of the present invention is prepared byimmunizing an animal with an immunogen comprising the new amino terminusof proteins cleaved during apoptosis and collecting antisera from thatimmunized animal. A wide range animal species can be used for theproduction of antisera. Typically an animal used for production ofantisera is a sheep, a donkey, a chicken, a rabbit, a mouse, a rat, ahamster, a goat, or a guinea pig. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (“KLH”) and bovine serum albumin (“BSA”). Otheralbumins such as ovalbumin, mouse serum albumin or rabbit serum albumincan also be used as carriers. Means for conjugating a polypeptide to acarrier protein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), carbodiimide andbis-diazotized benzidine.

Antibodies, both polyclonal and monoclonal, specific for epitopescomprising the new amino terminus of proteins cleaved during apoptosismay be prepared using immunization techniques, as are generally known tothose of skill in the art. A composition containing antigenic epitopesof the polypeptide of interest is used to immunize one or moreexperimental animals, such as a rabbit or mouse, which will then proceedto produce specific antibodies against the polypeptide. Polyclonalantisera may be obtained, after allowing time for antibody generation,by bleeding the animal and preparing serum samples from the whole blood,or in the case of chickens, by purifying the antibody from the eggs.

In preferred embodiments, the antibodies of the present invention arefurther purified by a process known as affinity purification. Methods ofaffinity purification of antisera, or other antibody compositions, arewell known to those of skill in the art. Generally, an antibodycomposition is subjected to chromatography wherein the antigen iscoupled to the resin of a column. Antibodies that are immunoreactivewith the antigen are retained in the column, whereas antibodies (andother proteins) that are not immunoreactive with the antigen flowthrough. The immunoreactive antibodies are then eluted from the column.Elution may be by a number of methods. One such method includes theaddition of 100% ethylene glycol to the column (Fornstedt, FEBS Lett177:195-199, 1984), while another is demonstrated in Example 1 herein.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen, as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster injection, alsomay be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immuneogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs. One of theaspects of the present invention is a polyclonal sera that is relativelyhomogenous with respect to the specificity of the antibodies therein.Typically, polyclonal antisera is derived from a variety of different“clones.” i.e., B cells of different lineage; mAbs, by contrast, aredefined as coming from antibody-producing cells with a common B-cellancestor, hence their “mono” clonality.

mAbs may be readily prepared through use of techniques such as thoseexemplified in U.S. Pat. No. 4,196,265. Typically, the production ofmAbs involves immunizing a suitable animal with a selected immunogencomposition, e.g., a purified or partially purified protein, polypeptideor peptide. The immunization composition is administered in a mannereffective to stimulate antibody producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep orfrog cells is also possible. The use of rats may provide certainadvantages (Goding, “Monoclonal Antibodies: Principles and Practice,”2^(nd) Edition, Academic Press, Orlando, Fla., pp. 60-74, 1986), butmice are preferred, with the BALB/c mouse being most preferred as thisis most routinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately about 5×10⁷ to about 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell line, generally one of thesame species as the animal that was immunized. Myeloma cell lines suitedfor use in hybridoma-producing fusion procedures preferably arenon-antibody producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cell lines may be used, as are known tothose of skill in the art (Goding, “Monoclonal Antibodies: Principlesand Practice,”2^(nd) Edition, Academic Press, Orlando, Fla., pp. 60-74,1986; Campbell, “Monoclonal Antibody Technology, Laboratory Techniques IBiochemistry and Molecular Biology,” Burden and Von Knippenberg, Eds.,Elsevier, Amsterdam, 13:75-83, 1984). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, X63-Ag8, 653, NS1/1, Ag4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul. Forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withhuman cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/O non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, Nature, 256:495-497, 1975; Eur. J. Immunoi., 6:511-519, 1976),and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, byGefter et al., Somatic Cell Genet., 3(2):231-236 (1977). The use ofelectrically induced fusion methods is also appropriate (Goding,“Monoclonal Antibodies: Principles and Practice,”2^(nd) Edition AcademicPress, Orlando, Fla., pp. 60-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to about 1×10⁻⁸. However, this does not pose a problem, asthe viable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture medium. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the medium is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium). See,e.g., Current Protocols in Immunology, eds. J. E. Coligan, et al, JohnWiley and Sons, Inc. 1992. Where azaserine is used, the medium issupplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective medium are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoor three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines. The clones can then bepropagated indefinitely to provide mAbs. The cell lines can be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific mAb produced by the fused cell hybrid. The bodyfluids of the animal, such as serum or ascites fluid, can then be tappedto provide mAbs in high concentration. The individual cell lines couldalso be cultured in vitro, where the mAbs are naturally secreted intothe culture medium from which they can be readily obtained in highconcentrations. mAbs produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography.

3. Polypeptides used to Generate Desired Antibodies

Polypeptides are disclosed herein as amino acid residue sequences. Thosesequences are written left to right in the direction from the amino tothe carboxy terminus. In accordance with standard nomenclature, aminoacid residue sequences are denominated by either a single letter or athree-letter code as indicated in Table 3 below.

TABLE 3 Amino Acid 3-Letter 1-Letter Residue Code Code Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T TrytophanTrp W Tyrosine Tyr Y Valine Val V

Modifications and changes can be made in the structure of a polypeptideof present invention and still obtain a molecule having likecharacteristics and function. For example, certain amino acids can besubstituted for other amino acids in a sequence without appreciable lossof receptor activity. Because it is the interactive capacity and natureof a polypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence (or, of course, its underlying DNA coding sequence)and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art (Kyte and Doolittle, J. Mol. Biol., 157:105-132,1982). It is known that certain amino acids can be substituted for otheramino acids having a similar hydropathic index or score and still resultin a polypeptide with similar biological activity. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. Those indices are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−0.3); proline (−1.6);histidine (−3.2); glummate (−3.5); asparatate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines that interaction of the polypeptide with othermolecules, such as enzymes, substrates, receptors, antibodies, antigens,and the like. It is known in the art that an amino acid can besubstituted by another amino acid having a similar hydropathic index andstill obtain a functionally equivalent polypeptide. In such changes, thesubstitution of amino acid whose hydropathic indices are within +/−2 ispreferred, those which are within +/−1 are particularly preferred, andthose within +/−0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. U.S. Pat. No. 4,554,101, incorporated hereinby reference, states that the greatest local average hydrophilicity of apolypeptide, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity and antigenicity, i.e. with abiological property of the polypeptide.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0 +/−1); glutamate (+3.0 +/−1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) proline (−0.5+/−1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithing +/−2 is preferred, those which are with in +/−1 are particularlypreferred, and those within +/−0.5 are even more particularly preferred.

As outlined above, amino acid substitution are generally therefore basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine (See Table 4, below). The present invention thus contemplatesfunctional or biological equivalents of a polypeptide comprising the newamino terminus of a protein cleaved by a caspase during apoptosis.

TABLE 4 Original Residue Exemplary Substitutions Ala Gly; Ser Arg LysAsn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg Met Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

4. Immunochemical Methods

The antibodies of the present invention will have particular use asreagents in immunochemical methods. Immunochemical methods include, butare not limited to, Western blotting, immunoaffinity purification,immunoprecipitation, ELISA, dot or slot blotting, RIA,immunohistochemical staining, immunocytochemical staining, and flowcytometry.

a. Flow Cytometry

The antibodies of the present invention may be used in methods of flowcytometry. Methods of performing flow cytometry to detect apoptosis arediscussed in Zhang et al., The Journal of Immunology, 157:3980-3987(1996) and Pepper et al., Leuk Res., 22(5):439-444 (1998). Generally,the cells are permeabilized to allow the antibody to enter and exit thecell. After permeabilization, the cells are incubated with an antibody.In preferred embodiments, the antibody is a monoclonal antibody. It ismore preferred that the monoclonal antibody be labeled with afluorescent marker. If the antibody is not labeled with a fluorescentmarker, a second antibody that is immunoreactive with the first antibodyand contains a fluorescent marker is used. After sufficient washing toinsure that excess or non-bound antibodies are removed, the cells areready for flow cytometry. Of course, the staining technique describedabove is also appropriate for the preparation of cells for other methods(e.g., fluorescent microscopy).

Using the antibodies of the present invention for flow cytometry,apoptotic cell would be indicated by an increase in the fluorescentintensity of the cell over control, non-apoptotic cells. The apoptoticcells may be sorted by their increase in fluorescence and subjected tofurther analysis (Zhang et al., The Journal of Immunology,157:3980-3987, 1996).

b. Immunoassays

Immunoassays of the invention include the various types of enzyme linkedimmunosorbent assays (ELISAs), as are known to those of skill in theart. However, it will be readily appreciated that other usefulembodiments include radioimmunoassays (“RIAs”) and other non-enzy mnelinked antibody binding assays and procedures.

U.S. Pat. No. 5,744,319 describes the use of antibodies to humantrypaste in what is commonly known as a double antibody-sandwich ELISA.The basic protocol for a double antibody-sandwich ELISA is as follows: Aplate is coated with antibodies (called capture antibodies) specific forthe antigen being assayed. The plate is then washed with a blockingagent, such as bovine serum albumin (BSA) to block nonspecific bindingof proteins (antibodies or antigens) to the test plate. The test sampleis then incubated on the plate coated with the capture antibodies. Theplate is then washed, incubated with detect antibodies, washed again,and incubated with a specific antibody-label conjugate. Afterincubation, the unbound conjugate is washed from the plate. The presenceof the bound antibody-label conjugate is indicated by detection of thelabel.

In preferred embodiments, the capture antibody is an anti-PARP antibody(i.e. to the about 89 kd fragment of PARP) of the present invention andthe detect antibody is an antibody that is immunoreactive with theregion of PARP corresponding to the carboxy terminal fragment ofcaspase-cleaved PARP but is not specific to the new amino terminus (suchas clone C2-10, Pharmingen; San Diego, Calif.; Cat.# 65196E). Of course,in light of the present disclosure many variations of a doubleantibody-sandwich ELISA will be apparent to those of skill in the art,including using the anti-PARP antibody of the present invention as thedetecting antibody or the detecting antibody may be labeled.

In other ELISAs, proteins or peptides are immobilized onto a selectedsurface, preferably a surface exhibiting a protein affinity, such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed material, one would then generally desire to bindor coat a nonspecific protein that is known to be antigenically neutralwith regard to the antibodies of the present invention, such as bovineserum albumin (BSA) or casein, onto the well. This allows for blockingof nonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antibodies ontothe surface. When the antibodies were created in an animal byconjugating a polypeptide to a carrier protein (e.g., KLH), it ispreferred that a protein different from the carrier protein be used as ablocking agent, because of the possibility of the presence of antibodiesto the blocking protein in the antibody composition.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with an antibodycomposition of the present invention in a manner conducive to immunecomplex (antigen/antibody) formation. Such conditions preferably includediluting the antibody composition of the present invention with diluentssuch as BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween® 20. These added agents also tend to assist in the reductionof nonspecific background. The layered antibody composition is thenallowed to incubate for, e.g., from 2 to 4 h, at temperatures preferablyon the order of about 25° C. to about 37° C. Following incubation, theantibody composition-contacted surface is washed so as to removenon-immunocomplexed material. A preferred washing procedure includeswashing with a solution such as PBS/Tween® 20, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the antibody composition of the present invention, and subsequentwashing, the occurrence and the amount of immunocomplex formation may bedetermined by subjecting the complex to a second antibody havingspecificity for the antibody of the present invention. To provide adetecting means, the second antibody will preferably have an associateddetectable label, such as an enzyme label or fluorescent molecule thatwill generate a signal, such as color development upon incubating withan appropriate substrate. Thus, for example, one will desire to contactand incubate the antibody-bound surface with a peroxidase-conjugatedanti-rabbit IgG for a period of time and under conditions that favor thedevelopment of immunocomplex formation (e.g., incubation for 2 h at roomtemperature in a PBS-containing solution such as PBS/Tween® 20). Thesecond antibody also may be conjugated to a hapten such as biotin thatcan be detected by avidin or streptavidin conjugated to an associated,detectable label.

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2.2′-azino-di-(3-ethylbenzthiazoline)-6 sulfonic acid (“ABTS”)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

A number of immunoassays are discussed in U.S. Pat. Nos. 5,736,348,5,192,660, and 4,474,892.

c. Western Blots

The compositions of the present invention will find use in immunoblot orWestern blot analysis. Methods of Western blotting are well known tothose of skill in the art and detailed methods are provided in theCurrent Protocols in Immunology (Id.). Generally, a protein sample issubjected to SDS-PAGE at such conditions as to yield an appropriateseparation of proteins within the sample. The proteins are thentransferred to a membrane (e.g., nitrocellulose, nylon, etc.) in such away as to maintain the relative positions of the proteins to each other.

In preferred embodiments, visibly labeled proteins of known molecularweight are included within a lane of the gel. These proteins serve as amethod of insuring that adequate transfer of the proteins to themembrane has occurred and as molecular weight markers for determiningthe relative molecular weight of other proteins on the blot.

Subsequent to transfer of the proteins to the membrane, the membrane issubmersed in a blocking solution to prevent nonspecific binding of theprimary antibody. In preferred embodiments, the primary antibody is anantibody to the new amino terminus of proteins cleaved during apoptosis.In more preferred embodiments, the antibody recognizes the new aminoterminus created by the cleavage of PARP by caspase 3 or caspase 7during apoptosis.

The primary antibody may be labeled and the presence and molecularweight of the antigen may be determined by detection of the label at aspecific location on the membrane. However, in preferred embodiments,the primary antibody is not labeled and the blot is further reacted witha labeled secondary antibody. This secondary antibody is immunoreactivewith the primary antibody. In preferred embodiments, the secondaryantibody is antibody to rabbit imunoglobulins and labeled with alkalinephosphatase (Promega, Madison, Wis.; Cat. # 5373B) or horseradishperoxidase.

An apparatus for, and methods of performing, Western blots are describedin U.S. Pat. No. 5,567,595.

d. Immunoprecipitation

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Methods ofimmunoprecipitations are described in U.S. Pat. No. 5,629,197.Immunopreciptitation involves the separation of the target antigencomponent from a complex mixture, and is used to discriminate or isolateminute amounts of protein. For the isolation of cell-surface localizedproteins, nonionic salts are preferred, since other agents, such as bilesalts, precipitate at acid pH or in the presence of bivalent cations.

5. Screening Assays

In yet another aspect, the present invention contemplates a process ofscreening substances for their ability to affect apoptosis. Utilizingthe methods and compositions of the present invention, screening assaysfor the testing of candidate substances can be derived. A candidatesubstance is a substance which potentially can promote or inhibitapoptosis within a cell sample contacted with the substance.

A screening assay of the present invention generally involvesdetermining the ability of a candidate substance to affect cellularprocesses leading to the production of an epitope that is immunoreactivewith an antibody composition of the present invention.

As is well known in the art, a screening assay provides a cell, or groupof cells, under conditions suitable for testing apoptosis. Theseconditions include pH, temperature, tonicity, and the presence ofrelevant factors involved in apoptosis (e.g., growth factors).

The pH is preferably from about a value of 6.0 to a value of about 8.0,more preferably from a value of about 6.8 to a value of about 7.8 and,most preferably a value of about 7.4. In a preferred embodiment,temperature is from about 20° C. to about 50° C., more preferably fromabout 30° C. to about 40° C. and, even more preferably about 37° C.Osmolarity is preferably from about 5 milliosmolar to about 400milliosmolar and, more preferably from about 200 milliosmolar to about400 milliosmolar and, even more preferably from about 290 milliosmolarto about 310 milliosmolar. The presence of factors can be required forthe proper testing of apoptosis in specific cells. Such factors include,for example, the presence or absence (withdrawal) of a growth factor,cytokine, such as an interleukin, colony stimulating factors, orneurotrophic factor.

a. Screening Assays for Compounds that Induce Apoptosis

The present invention provides a process of determining the presence ofan epitope produced in cells that are undergoing apoptosis, thus amethod of detecting apoptosis. Therefore, such a method may be utilizedto determine if a candidate substance is inducing apoptosis in abiological sample. A biological sample to be screened can be abiological fluid such as extracellular or intracellular fluid or a cellor tissue extract or homogenate. A biological sample can also be anisolated cell (e.g., in culture) or a collection of cells such as in atissue sample or histology sample. A tissue sample can be suspended in aliquid medium or fixed onto a solid support such as a microscope slide.

In accordance with a screening assay process, a biological sample isexposed to a candidate compound being assayed. Typically, exposure isaccomplished by contacting the biological sample with the candidatecompound. Of course, one may contact a large number of cells with thecandidate compound and a biological sample may be taken from thosecells. For example, one may administer the candidate compound to ananimal and collect a biological sample from the animal. Administrationmay be orally, transdermally, superficially, by inhalation, vaginally,retroductally (such as intraductal in mammary gland), intraveneously,intranasally, subcutaneously, rectally, or intermuscularly. Methods ofadministering compositions to animals by these routes are well known tothose of skill in the art.

The biological sample may be a blood sample or a tissue sample. Thetissue sample may be a biopsy, wherein the animal may not need to besacrificed prior to collection of the sample, or may be a tissue samplecollected from an animal following euthanasia or a sample collectedduring autopsy.

The biological sample is exposed to the candidate compound underconditions and for a period of time sufficient for induction ofapoptotic processes. Such conditions and time periods may be determinedby using compounds known to induce apoptosis in a given sample.Biological reaction conditions include ionic composition andconcentration, temperature, pH and the like. Ionic composition andconcentration can range from that of distilled water to a 2 osmolarsolution. Preferably, osmolarity is from about 100 milliosmolar to about400 milliosmolar and, more preferably from about 200 milliosmolar toabout 300 milliosmolar. Temperature preferably is from about 4° C. toabout 100° C., more preferably from about 15° C. to about 50° C. and,even more preferably from about 25° C. to about 40° C. pH is preferablyfrom about a value of 4.0 to a value of about 9.0, more preferably fromabout a value of 6.5 to a value of about 8.5 and, even more preferablyfrom about a value of 7.0 to a value of about 7.5. The only limit onbiological reaction conditions is that the conditions do not cause asignificant level of apoptosis in the absence of a candidate compoundbut do allow apoptosis in the presence of a known inducer of apoptosis.

Exposure time will vary inter alia with the biological conditions used,the concentration of compound and the nature of the sample (e.g., fluidor tissue sample). Means for determining exposure time are well known toone of ordinary skill in the art. Typically, where the sample is fluid,the exposure time is from about 10 minutes to about 200 minutes,although longer exposure times may be needed in some techniques (e.g.,neuronal growth factor (NGF) withdrawal). The presence of apoptosis inthe sample is detected by contacting the sample with the apoptosisspecific antibodies of the present invention and detecting the formationand presence of antibody-polypeptide conjugates. Means for detectingsuch antibody-antigen conjugates are disclosed herein. In oneembodiment, detection is accomplished by detecting an indicator affixedto the antibody. Exemplary and well known such indicators includeradioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), fluorescent labels, such asfluoroscein, rhodamine, or phycoerythrin, a second antibody or an enzymesuch as horseradish peroxidase, alkaline phosphatase, or luciferase.Means for affixing indicators to antibodies are well known in the art.Commercial kits are available for the purpose.

b. Screening Assay for Compounds that Inhibit Apoptosis

Similar to the above assay for screening compounds for their ability toinduce apoptosis, the present invention also provides methods forscreening compounds that inhibit apoptosis. Generally, such methodsinvolve subjecting a biological sample to conditions that induceapoptosis. These conditions may be conditions commonly known to induceapoptosis or may be conditions found to induce apoptosis by methodsdescribed herein. Such conditions include, but are not limited to, pH,temperature, tonicity, the presence of relevant factors involved inapoptosis (e.g., growth factors), or compounds capable of inducingapoptosis (e.g., anisomycin, etoposide, dexamethasone, valinomycin,merocyanine 540, or butyrate).

It is contemplated that the cells could be contacted with a candidatecompound suspected to inhibit apoptosis prior to, simultaneous with, orfollowing induction of apoptosis in the biological sample. In preferredembodiments of this screening assay, the cells are contacted with thecandidate compound following induction of apoptosis, and then screenedas follows:.

First, the biological sample is exposed to the candidate compound for aperiod of time sufficient for inhibition of apoptotic processes. Suchtime periods may be determined by using compounds known to inhibitapoptosis in a given sample. Exposure time will vary inter alia with thebiological conditions used, the concentration of compound and the natureof the sample (e.g., fluid or tissue sample). Means for determiningexposure time are well known to one of ordinary skill in the art.Typically, where the sample is fluid, the exposure time is from about 10minutes to about 200 minutes.

Then, the presence of apoptosis in the sample is detected by contactingthe sample with the apoptosis specific antibodies of the presentinvention and detecting the formation and presence ofantibody-polypeptide conjugates. Means for detecting suchantibody-antigen conjugates are disclosed herein. In one embodiment,detection is accomplished by detecting an indicator affixed to theantibody. Exemplary and well known such indicators include radioactivelabels (e.g., ³²P, ¹²⁵I, ¹⁴C), fluorescent labels, such as fluoroscein,rhodamine, or phycoerythrin, a second antibody or an enzyme such ashorseradish peroxidase, alkaline phosphatase, or luciferase. Means foraffixing indicators to antibodies are well known in the art. Commercialkits are available.

The ability to inhibit apoptosis is indicated by a reduced level ofantibody-polypeptide conjugates in the sample as compared to a sample,subjected to the same conditions and inducer of apoptosis as the testsample, that was not contacted with the candidate compound.

6. Kits

In another aspect, the present invention provides for kits for detectingthe presence of epitopes that are immunoreactive with the antibodies ofthe present invention. Such kits comprise a first container containing afirst antibody being an antibody of the present invention, with theantibody present in an amount sufficient to perform at least one assay.In a preferred embodiment, the first antibody is immunoreactive with thenew amino terminus produced by cleavage of PARP by a caspase duringapoptosis.

The assay kits of the invention may further comprise a second containercontaining a second antibody that immunoreacts with the first antibody.Preferably, the secondary antibody is conjugated with a label(enzymatic, fluorometric, radioactive, etc.). The secondary antibody maybe from essentially any animal including, but not limited to cow, goat,sheep, horse, rabbit, chicken, or donkey. In some preferred embodiments,the secondary antibody is a goat antibody that is imunoreactive withrabbit antibodies.

In another embodiment, the kit of the present invention may furthercomprise an antibody recognizing a proliferation marker (such asanti-PCNA or anti-Ki-67). Such kits would have particular utility intechniques for determining the status of a tumor sample. In anotherembodiment, the kits of the present invention firther comprise anantibody to a surface marker (such as antibodies to the CD antigens).Such kits would have particular utility in flow cytometry techniquesallowing one to determine the type of cells undergoing apoptosis in amixture of cell types.

In a preferred embodiment, the kits of the present invention furthercomprise other antibodies or reagents for detecting apoptosis. Otherantibodies and reagents for detecting apoptosis include, but are notlimited to, reagents for performing the TUNEL reaction (such as thereagents of the Apoptosis Detection System, Fluorescein andcolorimetric; Promega; Madison, Wis.; Cat.#G3250, G7360), Annexin V(Gatti et al., J. Histochem. Cytochem., 46(8):895-900, 1998), calcein(Gatti et al., J. Histochem. Cytochem., 46(8):895-900, 1998), andCaspACE™ reagents (Promega; Madison, Wis.; Cat.#G3540, G3751, G720).Such kits may contain a separate container means for each antibody orreagent or may optionally combine two or more reagents in one containermeans. Of course, when combining reagents, it is important that thecombination not affect the activity of either reagent, and it ispreferred that the combination is such that the ratio of the reagents isuseful in its intended assay.

In other embodiments, the kits of the present invention may includecompositions known to induce apoptosis such as anisomycin, etoposide,dexamethasone, valinomycin, merocyanine 540, or butyrate.

EXAMPLES

The following examples are included for illustrative purposes. It willbe appreciated by those of skill in the art that the techniquesdisclosed in the examples which follow represent techniques thatfunction well in the practice of the invention, and thus can beconsidered to relate to preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

PARP is an enzyme involved in DNA repair that is cleaved by caspaseactivity during the process of apoptosis (Kaufmann et al., CancerResearch, 53:3976-3985, 01993). PARP is an about 116 kd polypeptidepresent in about 10⁶ copies per somatic cell. Caspase cleavage of PARP(as well as other caspase substrates) results in the generation of anewly-formed unique amino terminus in apoptotic cells that normallywould not be present in non-apoptotic cells.

Caspase cleavage of PARP occurs at the carboxyl terminal side of theaspartate residue of the DEVD caspase recognition sequence between Asp214 and Gly 215 of PARP. This area of PARP is shown in FIG. 2. Caspasecleavage separates the amino terminal nick sensor region from thecarboxyl terminal catalytic domain. The cleavage results in an about 30kd amino terminal DNA binding domain and a about 85 kd carboxyl terminaldomain with basal PARP activity.

In Examples 1 and 6, below, antibodies to the new amino terminus createdby the cleavage of PARP during apoptosis were produced, usingpolypeptides with amino acid sequences identical to the first sequenceof amino acids at the amino terminus of SEQ ID NO:1. The polypeptideused in example 1 is a 7-mer, identified as SEQ ID NO:2. Two differentpolypeptides were used in Example 6, a 6-mer, identified as SEQ ID NO:3,and a 9-mer, identified as SEQ ID NO:4. The Examples below alsoillustrate methods of isolating and using the antibodies of theinvention to detect apoptosis in a variety of different samples.

Example 1

Production of Antibodies to the New Amino Terminus Created by Cleavageof PARP During Apoptosis, Using a 7-Mer Polypetide

A. Synthesis of 7-Mer Polypeptide and Conjugation to KLH

To create antibodies to the new amino terminus of PARP created bycaspase cleavage, a seven amino acid polypeptide corresponding to theamino terminal seven amino acids of the 85 kd PARP fragment (underlinedamino acids in FIG. 2 SEQ. ID NO:2) was synthesized and linked to a sixcarbon spacer arm and a C-terminal cysteine-amide to yield the moleculeof FIG. 3.

The molecule of FIG. 3 was conjugated, through the carboxy-terminalcysteine residue of the molecule to Keyhole Limpet Hemocyanin (“KLH”), acarrier protein. Prior to conjugation, KLH was activated by thefollowing procedure:

1. 350 mg of KLH was dissolved in 30 ml of H₂O.

2. 70 mg of MBS was dissolved in no more than 3 ml of dimethyl formamide(“DMF”).

3. The MBS solution was mixed with the KLH solution, and stirred for 30min.

4. 10% the resulting mixture was dialized in H₂O, to determine exactyield, and analyzed as follows:

a. The dialyzed material was dried down.

b. The mass of dialyzed material was measured, and multiplied by 10 toobtain the mass of the whole. The mass of dialyzed material wassubtracted from whole mass to determine the mass of the remaining 90%.

5. The remaining 90% of the mixture was dialyzed in Phosphate-BufferedSaline (“PBS”). This dialyzed material was aliquotted into 4 mg samplesbased on the following calculation:$\frac{{Fraction}\quad {Weight}}{{Fraction}\quad {Volume}} = {\frac{4\quad {mg}}{x\quad {ml}}.}$

6. 4 mg aliquots were lyophilized and frozen until needed.

7. When needed, a 4 mg aliquot of lyophilized sample was solubilized. 4mg of polypeptide was added, and the aliquot shaken for 2 hours.

B. Production of Antisera to the 7-mer Polypeptide

The resulting KLH-conjugated polypeptide was used to produce antisera tothe polypeptide in rabbits. Injection and recovery of antisera wasperformed as follows:

1. A rabbit was initially immunized with 400 μg of KLH-conjugatedpolypeptide suspended in Freunds Complete adjuvant

2. The rabbit was injected with a booster shot of 200 μg ofKLH-conjugated polypeptide suspended in Freunds Incomplete adjuvant 12weeks after the initial immunization

3. Antiserum was collected using two separate bleedings of the rabbit.One bleeding was 10 days after the boost. The other bleeding was 13 daysafter the boost.

C. Purification of Antibodies from the Antiserum

Following collection, the antisera were combined and affinity purified,according to the following procedure:

1. Coupling to Gel

a) Polypeptides and Sulfolink® gel (Pierce; Rockford, Ill.; Cat.#20401.)were allowed to come to room temperature.

b) A Tris/EDTA (50 mM Tris, 5 mM EDTA-Na, pH 8.5) buffer was prepared.

c) Disposable chromatography columns were each packed with anappropriate amount of Sulfolink® gel (about 2 ml of gel per eachcolumn).

d) Each column was equilibrated with 10 volumes of the Tris/EDTA buffer.

e) Caps on the top and bottom of each column were replaced.

f) 1 mg of polypeptide per 1 ml of gel in was dissolved in the Tris/EDTAbuffer and added to the gel in each column. (i.e., about 2 mg ofpolypeptide per column).

g) The gel and solution were mixed well, so that the bed of each columnbecame unpacked. The gel and solution were mixed on a shaker for 15minutes, being careful not to shake so vigorously that the beds wouldbreak.

h) The columns were allowed to incubate at room temperature for 30minutes.

i) The buffer was drained from each column and washed with 10 volumes ofTris/EDTA buffer.

2. Blocking Non-Specific Sites on Gel

j) Caps on the top and bottom of each column were replaced.

k) 1 ml of blocking buffer (50 mM cysteine solution in 50 mM Tris, 5 mMEDTA-Na, pH 8.5) per 1 ml of gel (usually 2 ml of buffer) was added toeach column.

l) The gel and solution were mixed so that the bed of each column becameunpacked. The columns were shaken on a shaker for 15 minutes, takingcare not to shake so vigorously that the beads would break.

m) The columns were allowed to incubate at room temperature for 30minutes.

n) The buffer was drained from each column, and the column washed with20 volumes of salt buffer (1 M NaCl).

o) The caps were replaced on the top and bottom of column. Azide bufferwas added to any column to be stored for later use, and the column wasrefrigerated in an upright position.

3. Antibody Binding

p) Each column was washed with 10 column volumes of phosphate buffer.

q) Up to 47 ml of serum was mixed with the gel in a 50 mL conicalcontainer.

r) The conical container was incubated at 4° C. in a styrofoam coolerovernight on a shaker.

4. Elution of Antibody

s) The gel and serum were poured back into the column. The flow-throughwas collected in a separate container, labeled, and stored in a coldroom (4° C.) for later use.

t) The remaining gel and serum were washed with 25 column volumes ofsalt buffer (about 0.5 M NaCl) to remove non-specific antibody.

u) The antibody was eluted from each column with glycine buffer. 15-20ml was collected in a 50 ml conical container, containing 1.1 mlTris-HCL pH 9.5. This is enough base to neutralize the 15-10 mL sample.

v) The eluted antibody was dialyzed in dialysis buffer (10 mM sodiumphosphate, 20 mM sodium chloride, pH 7.4) overnight with 1 change beforethe end of the day.

w) The dialyzed antibody was frozen until needed.

x) The column was stored by adding azide buffer thereto, andrefrigerating upright.

Example 2

Specific Recognition of the New Amino Terminus Created by Cleavage ofPARP During Apoptosis

The antibodies generated in Example 1 were used in a Western blotprocedure to demonstrate their ability to recognize the about 85 kdfragment of PARP and their inability to recognize uncleaved PARP.

A. Materials and Methods 1. Cleavage of PARP

Bovine spleen PARP (0.22 mg/ml; BIOMOL, Plymouth Meeting, Pa.; Cat#SE-165) was cleaved with recombinant CPP32 (Pharmingen International,San Diego, Cailf.; Cat# 66281T). In a tube, 104 μl of cleavage buffer(20 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS, 10%sucrose) (pH 7.2) were combined with 91 μl (20 μg) bovine spleen PARP,and 5 μl (1 μg) of recombinant CPP32. For efficient cleavage of PARP byCPP32, the tube was incubated at 37° C. for two hours. The reaction wasstopped by transferring to −20° C. Uncleaved PARP was obtained directlyfrom the stock tube for Western analysis.

2. Induction of Apoptosis in HL-60 Cells

Human promyelomonocytic leukemia cells, HL-60, were cultured inRPMI-1640 (Sigma; St. Louis, Mo.) and 10% fetal bovine serum (Hyclone)using standard tissue culture protocol. Log phase HL-60 cells werepelleted (350 g for 5 min) and resuspended in fresh medium at 5×10⁵cells/ml.

Apoptosis was induced in an aliquot of the cells by incubation in thepresence of anisomycin (2 μg/ml) (Sigma) for 2 hours at 37° C. To beused as a control for cells that were not induced into apoptosis, aseparate aliquot was incubated for 2 hours at 37° C. in the absence ofanisomycin. After the incubation period, the treated and untreated cellswere pelleted as before and washed twice with ice cold phosphatebuffered saline (PBS). Subsequently, the cells were resuspended in 1 mlPBS and transferred to a microcentrifuge tube, pelleted and resuspendedin PBS and 4×sample buffer (2.0 ml glycerol, 2.0 ml 10% SDS, 0.25 mgbromphenol blue, 2.5 ml stacking gel 4×buffer (6.06 g Tris-Base, 4 ml10% SDS, adjusted to pH 6.8 with concentrated HCl and brought to 100 mlwith H₂O, 4% DTT, H₂O to 10 ml) to a concentration of 4×10⁵ cells/20 μl.The samples (lysates) were then heated to 97° C. for 5 min and stored at−20° C until use.

3. Western Blot

Aliquots containing 125 ng of PARP from Tube A (full length PARP) andfrom Tube B (PARP cleaved by recombinant caspase 3) were combined with4×sample buffer (see above). The aliquots and the lysates from treatedand untreated HL-60 cells were heated for 3 min at 97° C. Samples werethen vortexed and centrifuged at 4000×g for 1 min. Samples were loadedon to a 4-20% SDS-PAGE pre-cast gel (Novex, San Diego, Calif.; Cat #EC60252) and the gel was run at constant current (30 mAmps) for 1 hr atroom temperature. The gel buffer was Laemmli buffer (25 mM Tris-base and192 mM glycine)+0.1% SDS.

The contents of the gel were then transferred to a nitrocellulose filterusing the BIORAD trans-blot system (Cat # 1620115). The transfer wasperformed at 100V for 1 hr at 4° C. in 20% methanol in Laemmli buffer(25 mM Tris-base, 192 mM glycine). Transfer was confirmed by Coomassieblue staining of the gel followed by destaining in 50% methanol/10%glacial acetic acid. Following transfer, the filter was stained with0.2% Ponceau S to visualize the individual lanes. The filter was thencut to allow incubation with different antibodies.

Prior to incubation with the 1° Abs, the filters were blocked with TBST(20 mM Tris-HCL pH 7.6; 150 mM NaCi; and 0.05% (v/v) Tween-20) plus 1%bovine serum albumin (Bayer, Fraction V; Cat#81-003-3) overnight at 4°C. The primary antibody (1:1000 dilution) was incubated with the filtersfor 2 hr at RT with oscillation. After the incubation period, thefilters were washed three times with oscillation for 15 min in TBST.Subsequently, the filters were incubated with the 2° Ab (donkeyanti-rabbit AP; Jackson Laboratories, West Grove, Pa.; Cat.#711-055-152; 0.6 mg/ml stock used at 1:5000 dilution) for 1 hr at roomtemperature with oscillation, followed by washing the filters threetimes for 10 min in TBST then washing in TBS (20 mM Tris-HCl, pH 7.6;150 mM NaCl).

To visualize the antibody-antigen complexes, the filters were developedwith Western Blue (Promega, Madison, Wis.; Cat #5384B) for 5-10 min inthe dark.

B. Specific Recognition of Cleaved PARP

To demonstrate that the antibodies generated in Example 1 were specificto the cleaved form of PARP, the antibodies were used in a Western blotprocedure on a gel containing PARP purified from bovine spleen. Tocreate cleaved PARP, the PARP purified from bovine spleen was cleavedwith recombinant CPP32. A 1:1000 dilution of the affinity purifiedantibodies failed to produce a signal in the lane containing uncleavedPARP. However, using these same antibodies, a band was easily visible inthe lane containing PARP cleaved with recombinant CPP32. When a 1:2000dilution of antibodies that recognize both cleaved and uncleaved PARPwas used (Boehinger Mannheim, Indianapolis, Ind.; Cat. # 1835238), asingle band corresponding to uncleaved PARP was visualized in theuncleaved sample, while two bands corresponding to uncleaved and cleavedPARP were visualized in the cleaved sample.

To demonstrate that the antibodies produced by the method of Example 1were specific to epitopes produced in apoptotic cells, protein lysateswere made from HL-60 cells treated with anisomycin to induce apoptosisand untreated HL-60 cells. A 1:1000 dilution of the affinity purifiedantibodies failed to produce a band in the untreated cell lane when usedin a Western blot, but was able to produce a band corresponding in sizeto the cleaved form of PARP in the treated cells. To ensure the presenceof the uncleaved form of PARP in both samples, a 1:2000 dilution of theantibodies that recognize both cleaved and uncleaved PARP was used.Uncleaved PARP was detected in lysates from both the untreated andtreated HL-60 cells, whereas the cleaved form is only detected in thesample from the treated cells.

Example 3

Immunohistochemical Analysis of Apoptotic Cells Using Antibodies to theNew Amino Terminus Created by Cleavage of PARP During Apoptosis

The antibodies produced according to Example 1 were used inimmunohistochemical analysis to detect cells undergoing apoptosis.

A. Materials and Methods 1. Generation of HL-60 Apoptotic Cell Smears

HL-60 cells were grown as described in Example 2. When the cells were ata concentration of about 3×10⁵ cells/ml, the cells were pelleted andresuspended in fresh RPMI 1640 with 10% fetal bovine serum at aconcentration of 5×10⁵ cells/ml. The cells were then split into twoaliquots (3 ml/aliquot). One aliquot, hereafter referred to as the“treated cells”, was incubated with anisomycin (2 μg/ml) for 1.5 hr at37° C. and 5% CO₂. The other aliquot, hereafter referred to as the“untreated cells”, was incubated for 2.5 hr at 37° C. and 5% CO₂ in theabsence of anisomycin. After the incubation, the cells were pelleted (5min, 400×g), washed twice with DPBS (Sigma; St. Louis, Mo.; Cat.#D-8537) and pelleted, and each pellet was resuspended in 5 ml DPBS and 5ml MonoSol fixative from a MonoPrep 2 starter kit (MonoGen, Inc.;Herndon, Va.: Cat.# SK210). The cells were diluted to 10⁵/ml using 1:1DPBS and MonoSol and 10⁶ (10 ml) of the cells were added to MonoPrep“collection vials”.

The cell preps were processed onto microscope slides using the MonoPrep2standard slide Preparation Procedure for Monolayer Slide Preparation.Briefly, cells were drawn from the collection vials onto the filters(Ultraclean Filter Assemblies) using the syringe slide filter assembly.The filters were transferred (cell side down) to Fisher Super Frost Plusslides (Fisher Scientific; Pittsburg, Pa.; Cat# 12-550-15) usingforceps. Five drops of MonoFix reagent were added to the top of eachfilter for 2 min. Several layers of folded Kimwipe were placed on top ofthe filters and the cells were transferred to the slides by pressingfirmly. The filters were allowed to dry completely for several minutes.The filters were removed with forceps and discarded.

Two of the slides (one control and one anisomycin treated) were fixedfor 30 min in 4% paraformaldehyde in a humidified plastic slide rack.The slides were rinsed in DPBS and tranferred to a glass coplin jarcontaining DPBS and stored at 4° C. Another two slides (one control andone anisomycin treated) were fixed by immersing in acetone for 5 min ina Coplin far. The slides were air dried in a chemical fume hood for 5-10min then transferred to a Coplin jar containing DPBS and stored at 4° C.

2. Immunohistochemical Staining of HL-60 Cell Smears

The fixed cell smears were treated with 0.3% H₂O₂/DPBS for 3 min. Theslides were blocked using 5% horse serum in PBS for 15 min, then as muchliquid as possible was removed without allowing the slides to dry.Affinity purified anti-PARP fragment antibody was diluted 1:50 in DPBSto a final concentration of 10.88 μg/ml and added to each slide for45-60 min in a plastic slide rack with humid atmosphere to keep thesamples from drying.

Following two 10 min washes with DPBS, a 1:1000 dilution of a biotinconjugated AffiniPurified Donkey Anti-rabbit IgG (H+L) (Jacksonlaboratories, Inc.; West Grove, Pa.; Cat# 711-065-152) was added to eachslide for 1 hr. Again the slides were washed twice in DPBS for 10 mineach wash.

Horseradish peroxidase conjugated avidin-biotin complex (VectastainElite ABC Kit PK6100 Standard; Vector Laboratories; Burlington, Cailf.)reagent was prepared by adding 50 μl Reagent A and 50 μl Reagent B to 5ml DPBS in a dropper bottle. An adequate amount of reagent to cover thecells on the slides was added to each slide and the slides wereincubated for 25 min.

Freshly prepared DAB solution (Zymed, South San Francisco, Calif.; BrdUstaining Kit; Cat# 93-3943) substrate solution was added to the slidesfor 10 min. DAB substrate solution was prepared by adding 50 μl20×Buffer concentrate to 950 μl water, then 50 μl H₂O₂, then 50 μl DABwere added to the slides.

Following washing twice with DPBS for 10 min, 1-2 drops of glycerolbased mounting medium were added on the cells and Corning #1 22×30 mmcoverslips were added on top.

B. Immunohistochemical Staining is Selective to Apoptotic Cells

In the paraformaldehyde fixed samples, 63% of anisomycin treated cellswere stained with anti-PARP antibodies, whereas only 16% of theuntreated cells were stained. In the acetone fixed samples, 72% of theanisomycin treated cells were stained, whereas only 4% of the untreatedcells were stained. The stained cells in the untreated samples tended tohave an apoptotic (round distinct) morphology and are likely torepresent cells undergoing apoptosis in the absence of anisomycintreatment.

Example 4

Comparison of PARP Antibody Staining to TUNEL

In order to compare using anti-PARP antibodies to the TUNEL assay todetect apoptotic cells, the TUNEL assay was performed followed byimmunocytochemical staining with anti-PARP antibodies on the same HL-60cell smear.

A. TUNEL Assay

Treated HL-60 cell smears were created as described in Example 3. Thecells were permeabilized in 0.2% Triton X-100/PBS for 5 min at roomtemperature, followed by three, 5 min washes in PBS. The smears werethen incubated in equilibation buffer (200 mM potassium cacodylate, pH6.6; 25 mM Tris-HCl, pH 6.6; 0.2 mM DTT; 0.25 mg/ml BSA; 2.5 mM cobaltchloride) for 5 min at room temperature. TdT mix (50 μl equilibrationbuffer, 1 μl fluorescein-12-dUTP, and 1 μl of TdT enzyme; Promega;Madison, Wis.; Cat#G3250) is then added to the slides and incubated for1 hr at 37° C., followed by a rinse in 2×SSC for 15 min at roomtemperature, and three washes for 5 min in PBS. All subsequent stepswere done in the dark to protect the fluorescein signal.

B. Immunocytochemistry

Following the TUNEL assay, the smears were stained with the anti-PARPantibodies. Briefly, the smears were blocked with 1% BSA, 2% horseserum, and 10 μg/ml donkey IgG in PBS, followed by a wash with PBS. Thesmears were then incubated overnight at 4° C. with a 1:100 dilution (6.7μg/ml) of affinity purified anti-PARP PARP antibodies that werepreabsorbed with HL-60 cells. The preabsorption was performed byincubating the antibody with lysates of untreated HL-60 cells that werefixed in 10% formalin and rinsed in PBS.

After the overnight incubation, the smears were washed four times withPBS, followed by incubation with 0.3% hydrogen peroxide for 3-5 min atroom temperature. The smears were washed three times for 5 min per wash,then incubated for 70 min at room temperature with a 1:500 dilution ofdonkey anti-rabbit-biotin secondary antibody (Jackson Laboratories;Cat#711-065-152). After three, 5 min washes in PBS, the smears wereincubated with 100 μl Streptavidin-HRP (Zymed) for 30 min at roomtemperature. Following three more washes, DAB reagent (Zymed;Cat#00-2114) was added to the smears. Development was at roomtemperature for 10 min and was stopped by rinsing with water. Prior toanalysis, the smears were mounted in Vectashield.

C. Analysis

Comparison of the same field of cells using fluorescent (TUNEL) andlight microscopy allowed comparison of the two techniques. In thetreated HL-60 cell sample, approximately 90% (37/41) of the TUNELpositive cells were scored as also staining with the anti-PARP antibodyimmunocytochemistry procedure. Interestingly, a considerable number ofcells in the treated sample were stained by the anti-PARP antibodyimmunocytochemistry procedure but were negative by the TUNEL assay. Thisresult may be attributed to the ability of the anti-PARP antibodyimmunocytochemistry procedure to detect an apoptotic step (caspaseproteolysis) that is prior to DNA fragmentation. Thus, anti-PARPantibody immunocytochemistry procedure may be a more sensitive method ofdetecting the early events of apoptosis than the TUNEL assay.

Example 5

Preparation of Antibodies to the New Amino Terminus Created by Cleavageof PARP During Apoptotis, Using a 6-Mer Polypeptide and a 9-MerPolypetide

6-mer and 9-mer polypeptides each having a sequence corresponding to theamino terminal 6 or 9 terminal amino acids of the about 85 kd PARPfragment, shown in FIG. 2), were synthesized and linked to a six carbonspacer arm, a 6-aminohexanoic acid group, and a C-terminal cysteine toyield the two molecules shown below:

Formula 1: Gly-Val-Asp-Glu-Val-Ala-[6 Aminohexanoic acid]-Cys

Formula 2: Gly-Val-Asp-Glu-Val-Ala-Lys-Lys-Lys-[6 Aminohexanoicacid]-Cys

The amino acid sequence of the 6-mer polypeptide of Formula 1, i.e.GVDEVA, is identified herein as SEQ ID NO:3. The amino acid sequence ofthe 9-mer polypeptide of Formula 2, i.e. GVDEVAKKK, is identified hereinas SEQ ID NO:4.

The preparation of Formula 1 was determined to be 93% pure by HPLCanalysis, and the preparation of Formula 2 was 96% pure. Bothpreparations were found to be soluble in water. Each polypeptide wasconjugated to KLH through the terminal cysteine residue of eachformulation, according to the conjugation procedure of Example 1.A. Theresulting conjugated polypeptides were used to produce antisera to eachpolypeptide, in rabbits, according to antibody production procedure ofExample 1.B. Specifically, each conjugated polypeptide was injected intoa rabbit, the rabbit was boosted two weeks later and the antiserum usedin these studies was obtained three weeks after the second boost andstored at 4° C.

Example 6

Purification of Anti-PARP 6-Mer and Anti-PARP 9-Mer Antibodies fromRabbit Antiserum

A. Preparation of Resins

Three milligrams of the 6-mer and 9-mer polypeptides produced andcharacterized as described in Example 5, above, were dissolved inseparate 3 ml aliquots of TE (50 mM Tris, 5 mM EDTA pH 8.5).

Three milliliters of Sulfolink™ resin (Pierce, 20401) were placed into a15 ml column for each polypeptide and washed with 30 ml of TE solution.The polypeptides were added to each column and shaken gently,end-over-end, for 15 minutes, then allowed to stand at room temperaturefor 30 minutes. The columns were then each washed with 30 ml TEsolution. Then 3 ml of TE containing 50 mM cysteine (Sigma Corp., C7755)added to each column and the columns were shaken gently, end-over-end,for 15 minutes and allowed to stand at room temperature for 30 minutes.Then each of the columns was washed with 30 ml water and storedovernight at 4° C.

B. Purfications of Antibodies

Ten milliliters of rabbit antiserum from rabbits innoculated with the6-mer polypeptide conjugate prepared as described in Example 5 was addedto 10 ml PBS and mixed gently. The resin was then removed from thecolumn and washed with 30 ml PBS, drained, and the resin was added tothe serum. The serum/resin mixture was shaken gently, end-over-end, forone hour. The above steps were repeated for the rabbit antiserum fromrabbits innoculated with the 9-mer polypeptide prepared as described inExample 5. The serum/resin mixtures were poured into a column and washedwith 30 ml PBS until the OD280 of the eluant was less than 0.01. Theantibody from each column was eluted with successive 3 ml aliquots of0.1 M glycine pH 3.1. Affinity purified anti-PARP (9-mer) was collectedin five fractions, affinity purified anti-PARP (6-mer) was collected inthree fractions. The fraction tubes each contained 0.3 ml 2 M Tris pH8.0. The fractions for each antibody were pooled and the concentrationdetermined by spectrophotometric reading at OD280. Each antibody wasdialyzed against PBS 4° C. The 9-mer polypeptide conjugate yielded 15 mlof 0.327 mg/ml anti-PARP (9-mer) for a total of 4.9 mg. The 6-merpolypeptide conjugate yielded 10 ml of 0.21 mg/ml anti-PARP (6-mer) fora total of 2.1 mg.

Example 7

Immunocytochemistry Analysis of Apoptotic Jurkat Cells Using AffinityPurified Anti-PARP Antibodies

Jurkat cells were suspended in fresh medium at a concentration of 5×10⁵cells per milliliter. Twelve milliliter cultures of the cells weremaintained in suspension culture in each of two T-75 flasks. Apoptosiswas induced in the cells by treating them with monoclonal anti-Fasantibody (PanVera Corp., SY-001) at a final concentration of 100 ng/mlfor 3 hours at 37° C. in 5% CO₂. The cells were then harvested bycentrifugation at 250×g for 5 minutes. The supernatant was then removedand the cell pellet washed with 7 ml of PBS (Sigma D-8779) andresuspended to 1.5×10⁶ cells per milliliter in PBS.

Poly-1-lysine (Sigma P8920) was diluted in water to 0.1 mg/ml. Then 10μl of the poly-1-lysine solution was added to each well of an 8 wellslide (Cel-Line corporation) and the drop dispersed to cover the entirearea of the well. The slides were then air-dried at room temperature forabout 45 minutes. The slides were then washed by dipping them severaltimes in water and allowing them to air dry.

The cell suspension was thoroughly mixed and 15 μl added to each well ofthe slide. The cells were allowed to settle and attach for 5 minutesprior to placing them directly in 10% buffered formalin (Fisher Corp.,23-245684) and incubated at room temperature for 25 minutes. The slideswere then washed by dipping them twice in coplain jars containing TBS(Promega Corp., AA640). The cells were then observed to be attached andthe slides stored in TBS at 4° C. until used.

The following immunocytochemistry analysis was then performed on threeof the apoptosis-induced Jurkat cell slides and three untreated,control, Jurkat cell slides. The six slides were permeabilized bysoaking in a coplain jar containing PBS with 0.2% Triton X-100 for fiveminutes at room temperature. They were then washed three times, fiveminutes each in a coplain jar containing PBS. They were then blocked forone hour at 37° C. in PBS containing 5% horse serum (Hyclone,SH30074-03) and 0.1% Tween-20 (Sigma, P1379). The primary antibodies(from Example 1) were diluted into the same blocking buffer and about 40μl added per well. The antibodies and concentrations used are listedbelow. The slides were then placed horizontally into a humidifiedchamber and incubated overnight at 4° C.

TABLE 1 ANTIBODY FINAL CONCENTRATIONS TESTED anti-PARP 6-mer none, 1.25,2.5, 5.0 μg/ml anti-PARP 9-mer none, 1.25, 2.5, 5.0 μg/ml anti-PARP7-mer none, 1.25, 2.5, 5.0 μg/ml

The slides were then washed in a coplain jar four times for 10 minuteseach in PBS containing 0.1% Tween-20. This was followed by two washesfor 10 minutes each in PBS alone. The slides were drained and 40 μldonkey anti-rabbit Cy3 conjugated antibody (Jackson Labs, #711-165-152,stock 0.625 mg/ml in 50% glycerol), diluted 1:250 in block buffer wasadded. The slides were incubated, protected from light, for two hours atambient temperature in a humidified chamber. The slides were then washedtwice for 5 minutes each in PBS, once for 5 minutes in PBS containing0.1% Tween-20, and lastly for 5 minutes in PBS. The wells were mountedusing mounting medium containing DAPI (Vector Labs, H1200) and #1coverslip. The fluorescence was observed with a Zeiss Fluorescentmicroscope with a 63×objective and rhodamine/DAPI filter cube. Digitalphotos were taken and analyzed.

The resulting photographs demonstrated all three antibodies tested hadpreferential reactivity with apoptotic cells. The 7-mer antibody had nobackground staining on the negative control slides and it also had thestrongest apoptosis-specific reactivity. It was optimally reactive at1.25 μg/ml. The anti-PARP (9-mer) and anti-PARP (6-mer) antibodypreparations had low amounts of background reactivity, but did producefaint staining of non-apoptotic cells. Both were optimally reactive at1.25 μg/ml.

Example 8

Western Blot Analysis Using Anti-PARP (6Mer) and Anti-PARP Anti-PARP(9-Mer) Antibodies

In this Example the purified antibodies described in Example 6 were usedfor Western blot analysis. PIPES buffer was prepared to contain thefollowing components: 20 mM PIPES, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS,10% sucrose, 70 ml DI water. The buffer was pH adjusted to pH 7.2 andthen brought to a final volume of 100 ml with DI water. The buffer wasfiltered through a 0.2 micron filter and stored sterile at 4° C. DTT wasadded just prior to use to a final concentration of 10 mM DTT

To prepare caspase-3 cleaved PARP antigen, in a 1.5 ml conical tube werecombined 2 μg Caspase-3 enzyme (Pharmingen, 66281U), 20 μg uncleavedbovine PARP (Biomol, SE165) and 99 μl PIPES buffer. This will yield 100μg/ml of PARP at a 10:1 substrate:enzyme ratio. This solution wasincubated at 37° C. for two hours with shaking three or four timesduring this period to keep the reactant suspended. The reaction wasstopped by transferring the solution to −20° C.

The samples were prepared to be run on a 4-20% SDS PAGE gel (Novex) asshown in Table 2, below.

TABLE 2 2X Sample load Sample ng/well stock TBST Buffer with DTT perwell Uncleaved 200 4.6 μl 45 μl 25 μl 20 μl Bovine PARP Cleaved PARP 201 μl 49 μl 50 μl 20 μl SeeBlue MW 20 μl 10 μl Markers

All the samples were heated at 95° C. for 5 minutes and the gel loadedand run. The proteins run into the gel were then transferred ontonitrocellulose by standard Western blot method. The nitrocellulosemembrane was blocked in TBST+1% BSA (Bovine Serum Albumin) at roomtemperature for one hour. A similar set of the three lanes on themembrane were incubated with each primary anti-PARP antibodypreparations (Example 5) diluted 1:5000 in TBST. They were incubatedwith shaking for one hour at room temperature and then the membraneswere washed three times for five minutes each in TBST. Donkeyanti-rabbit IgG AP conjugate (Promega Corp.) was diluted 1:5000 in TBST.The membranes were incubated in this secondary antibody for one hour atroom temperature and then washed four times in TBST for 5 minutes perwash. The membranes were incubated in Western Blue Stubstrate (PromegaCorp.) for up to four minutes to detect the bands.

Both anti-PARP antibodies detected the cleaved PARP on the Western blotwith the bands developing on the blot within about two minutes for theanti-PARP (9-mer) antibody and about four minutes for the anti-PARP(6-mer) antibody.

Example 9

Purification of Anti PARP (6-Mer) and Anti-PARP (9-Mer) Antibodies

This example uses ammonium sulfate precipitation, followed by affinitypurification to isolate the anti-PARP (6-mer) and anti-PARP (9-mer)antibodies from rabbit antiserum. Four milliliters of antiseracontaining antibody specific to the 6-mer polypeptide described inExample 5 were combined with 0.2 ml 5% dextran sulfate (Sigma D5251),and 0.36 ml 1 M CaCl₂ (Sigma C3881). The solution was mixed and thenincubated at room temperature for 30 minutes. Four milliliters ofantisera containing antibody specific to the 9-mer polypeptide describedin Example 5 were likewise treated. The solutions were then centrifugedat 14,000 rpm in a microcentrifuge at 4° C. The supernatants were theneach brought to 50% saturation with granular ammonium sulfate (SigmaA6387) and incubated overnight at 4° C. The solutions were thentransferred to 1.5 ml tubes and centrifuged at 14,000 rpm for 25 minutesat 4° C. The resulting antibody pellets were resuspended in a total of 2ml TBS for each antibody. Sodium azide was added to a finalconcentration of 0.02% and the solutions were stored at 4° C. untilused.

A. Preparation of Affinity Resin

All reagents were brought to room temperature prior to use. Twomilliliters of gel slurry (SulfoLink™ resin, Pierce #20401) were placedin a 5 ml polypropylene column (Schleicher & Schuell, 77227). This makesabout a 1 ml resin bed when settled. A medium size frit was used on thebottom of the column. The column was allowed to drain by gravity andthen equilibrated with 12 ml SulfoLink™ coupling buffer (Pierce,1852080) in 50 mM Tris pH 8.5, 5 mM EDTA. One milligram of eachpolypeptide, 6-mer and 9-mer, was added to 2 ml coupling buffer and theneach added to a separate 1 ml resin bed. The columns were mixed gentlyby rocking on a platform rocker for 15 minutes at ambient temperature,protected from the light. They were then incubated stationary for anadditional 30 minutes at room temperature. The columns were then drainedand washed with 6 ml coupling buffer.

B. Block Column Sites

A 7.9 mg/ml cysteine-HCl (Aldrich, C12,108-0) solution was prepared incoupling buffer. Two milliliters of this solution were added to theresin bed of each of the two columns. The columns were mixed gently byrocking on a platform rocker for 15 minutes at room temperature and thenfurther incubated stationary for 30 minutes at room temperature. Thecolumns were drained and washed with 16 ml of 1 M NaCl, followed by an 8ml water wash. Sodium azide was added to a final concentration of 0.02%in the final water wash and the columns stored at 4° C.

C. Affinity Purification of Antisera

The resin bed of each column was divided in half and 0.5 ml of the resinbed was used for each purification. All reagents were brought to ambienttemperature prior to use. The 0.5 ml resin in the column was washed with10 ml of TBS by gravity and allowed to drain. The bottom of each columnwas then sealed. Two ml of the TBS/antisera solution were added to theresin. The caps were added to seal the columns and then they were rockedgently for one hour at ambient temperature. The columns were placed instands, drained, and each washed with 5 ml TBS. The columns were theneach washed with 5 ml TBS/0.5 M NaCl. The antibody on each column waseluted into 1.5 ml eppendorf tubes, each containing 20 μl of 2 M Tris pH8.0 with 4 drops in each fraction (200 μl) of 0.1 M glycine pH3.0 (SigmaG7126). Each fraction was mixed as it was eluted to neutralize it. Atotal of about 18 fractions were taken. The protein elution wasmonitored with Pierce Coomassie Plus (Pierce, 18526210) by adding 5 μlsample to 100 μl reagent. They were mixed and the absorbance at 600 nmon a Dynatech MR5000 plate reader was recorded. The fractions withsignificant absorbance change were pooled for each antibody.

The solutions were then dialyzed using Pierce Slide-A-Lyzer™ 10,000molecular weight cutoff cassettes (Pierce, 55426) against PBS at 4° C.First one liter of PBS was used for 3 hours, this was then replaced withfresh PBS and the dialysis continued overnight at 4° C. Absorbancereadings were taken at 280 nanometers (A₂₈₀) directly, without dilution,on a Beckman DU600 under UV light. Each sample was blanked against PBS.Sodium azide was added to each solution, for a final concentration of0.02%. The resulting samples were stored at 4° C. until used.

The final yields were 0.44 mg for the anti-PARP (6-mer) antibody and0.845 mg anti-PARP (9-mer) antibody.

Example 10

Immunohistochemistry Analysis Using the Purified Anti-PARP Antibodies

This example compared the antibody preparations purified in Examples 1and 6 antibody preparations purified in Example 9 in animmunohistochemistry on using control and apoptotic, Jurkat cell slidesprepared as described in 7.

Five slides containing control, untreated Jurkat cells and five slidescontaining Jurkat cells were permeabilized by soaking them in a coplainjar containing PBS+2% Triton X-100 (Sigma, T9284) for 5 minutes at roomtemperature. They were then washed three times for five minutes each incoplain jars containing PBS. They were blocked in PBS+5% horse serum(Hyclone, SH30074-3) for 90 minutes at 37° C. in a 5% CO₂ environment.

Primary antibodies were diluted into blocking buffer as listed in thetable below. About 40 μl of each diluted antibody were added to eachwell. The slides were placed horizontally into a humidified chamber forone hour and then transferred to 4° C. and cubated overnight.

TABLE 3 Slide Antibody Concentration of Antibodies Used 1 Example 9anti-PARP (6-mer) none, 0.1, 0.5, 2.0 μg/ml 2 Example 9 anti-PARP(9-mer) none, 0.1, 0.5, 2.0 μg/ml 3 Example 6 anti-PARP (6-mer) none,0.1, 0.5, 2.0 μg/ml 4 Example 6 anti-PARP (9-mer) none, 0.1, 0.5, 2.0μg/ml 5 Example 1 anti-PARP (7-mer) none, 1.25, 2.5 μg/ml

The slides were then washed in coplain jars four times, 10 minutes each,in PBS+0.1% Tween-20. Then followed by two washes, 10 minutes each, inPBS alone. The slides were then drained and the secondary antibody wasadded. The secondary antibody was Donkey-anti-rabbit, Cy-3 conjugated,diluted 1:250 in blocking buffer (Jackson Immuno. Research Laboratories,Inc., West Grove, Pa., 711-165-152). The slides were then incubated fortwo hours at room temperature, protected from light. They were thenwashed in coplain jars protected from light. They were washed twice, 5minutes each, in PBS, followed by one 5 minute wash in PBS+0.1% Tween-20and a final 5 minute wash in PBS. The slides were then drained andmounted using mounting medium containing DAPI (35 μl/slide; Vector Labs,H-1200) and a #1 coverslip.

The fluorescence was observed with a Zeiss, fluorescent microscope,63×objective. Digital photos were taken with a Diagnostic Spot 2 camera.

While the anti-PARP (9-mer) antibody prepared in Example 6preferentially reacted with the apoptotic cells, there was highbackground staining levels in the control cells. Staining with thisantibody was optimal at the 0.5 μg/ml concentration. The anti-PARP(9-mer) antibody prepared in Example 9 had little, if any, staining ofthe control Jurkat cells and intense staining of apoptotic cells at the0.5 μg/ml concentration. The anti-PARP (6-mer) antibody prepared inExample 9 preferentially reacted with the apoptotic cells and there wasminimal staining in the control wells. The anti-PARP (6-mer) antibodyprepared in Example 6 resulted in staining of the apoptotic cells, butno staining in the control wells.

The antibody reactive against the 7-mer polypeptide (described inExample 1), provided the most intense signal at the lowest concentrationof all the preparations of either of the other two anti-PARP antibodiestested herein (i.e., anti-PARP (6-mer) or anti-PARP (9-mer) antibody).The antibody reaction against the 7-mer consistently provided the mostintense signal, and the signal was specific for apoptotic cells withlittle, if any background signal from staining of the control cells. Theoptimal concentration for this antibody was 1.25 μg/ml, while theoptimal concentration of anti-PARP (6-mer) and anti-PARP (9-mer)antibody preparations was 2.5 μg/ml.

Example 11

Immunocytochemistry Analysis Using Hybridoma Supernatant on ApoptoticJurkat Cells

Hybridoma supernatant was prepared by Harlan Corp. (Madison, Wis.) usingthe 7-mer polypeptide as described in Example 1. The culture supernatantwas contained in DMEM+20% fetal calf serum+HT. For this experiment,undiluted supernatant was used as the source for the dilutions. The Shammedium (no cells) consists of DMEM+20% FCS+HT. The control and anti-Fasantibody induced apoptotic Jurkat cells used in this Example wereprepared as described in Example 2 and stored at 4° C. in TBS untilused. Three slides of control cells and three slides of apoptotic cellswere used. The antibody dilutions used are listed in Table 4, below.

TABLE 4 1. Hybridoma supernatant none, 1:2, 1:5, 1:15 2. Sham mediumcontrol (negative control) none, 1:2, 1:5, 1:15 3. Rabbit anti-PARPantibody (7-mer) none, 1.25 μg/ml, 2.5 μg/ml (positive control)

The cells on the slides were permeabilized by soaking the slides in acoplain jar containing PBS+0.2% Triton X-100 for 5 minutes at ambienttemperature. The slides were then washed three times, five minutes each,in coplain jar containing PBS. The slides were blocked using about 35 μlper well of PBS+5% horse serum +0.1% Tween-20. They were blocked in thissolution for 90 minutes at 37° C. in a 5% CO2 environment.

The excess was then shaken off and 35 μl of the primary antibody dilutedin the blocking buffer as described above, were added per well. Theslides were then placed horizontally into a humidified chamber andincubated overnight at 4° C.

The slides were then washed in a coplain jar four times, ten minuteseach, in PBS+0.1% Tween-20, followed by two washes, 10 minutes each, inPBS alone. The slides were then dried and 35 μl of the appropriate Cy3conjugated secondary antibody, diluted 1:250 blocking buffer, were addedper well. Donkey-anti-mouse H+L, Cy3, secondary antibody (Jackson Labs,715-165-150) was used for the slides previously incubated with thehybridoma culture supernatant or the sham medium. Donkey-anti-rabbitH+L, Cy3, secondary antibody (Jackson Labs, 11-165-152) was used for theslides previously incubated with the positive control, rabbit antisera.The slides were then incubated for 2 hours at ambient temperature,protected from the light.

The slides were then washed in a coplain jar protected from light twice,5 minutes each, in PBS, then 5 minutes PBS+0.1% Tween-20, and a final 5minute wash with PBS. The slides were then drained and mounted with #1coverslips using about 35 μl mounting medium which contained DAPIcounterstain (Vector Labs, H-1200).

The fluorescence was observed with a Zeiss fluorescent microscope,63×objective. Digital photos were also taken and analyzed.

The hybridoma supernatant reacted specifically with the apoptotic cellsat an optimal dilution of 1:5. The 1.25 μg/ml concentration of the 7-merantibody reacted specifically with the apoptotic cells and the shammedium control had no resulting fluorescence. The hybridoma supernatantat 1:4 and 1:20 dilution also reacted positively to 100 ng caspase3-cleaved PARP in a Western blot analysis performed as described inExample 3 with the exception that the secondary antibodies listed abovewere used at a 1:10,000 dilution.

5 1 30 PRT Homo sapiens 1 Gly Val Asp Glu Val Ala Lys Lys Lys Ser LysLys Glu Lys Asp Lys 1 5 10 15 Asp Ser Lys Leu Glu Lys Ala Leu Lys AlaGln Asn Asp Leu 20 25 30 2 7 PRT Homo sapiens 2 Gly Val Asp Glu Val AlaLys 1 5 3 6 PRT Homo sapiens 3 Gly Val Asp Glu Val Ala 1 5 4 9 PRT Homosapiens 4 Gly Val Asp Glu Val Ala Lys Lys Lys 1 5 5 49 PRT Homo sapiens5 Lys Lys Gln Leu Pro Gly Val Lys Ser Glu Gly Lys Arg Lys Gly Asp 1 5 1015 Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys Lys Ser Lys Lys Glu 20 2530 Lys Asp Lys Asp Ser Lys Leu Glu Lys Ala Leu Lys Ala Gln Asn Asp 35 4045 Leu

What is claimed is:
 1. A composition comprising an antibodyimmunoreactive with a neoepitope produced in a cell undergoingapoptosis, said neoepitope comprising an amino terminus produced bycleavage of a protein at a cleavage site cleaved by a protease duringapoptosis, wherein said antibody is not immunoreactive with the proteinwhen not cleaved by the protease.
 2. The composition of claim 1, whereinthe antibody is a polyclonal antibody.
 3. The composition of claim 1,wherein the antibody is a monoclonal antibody.
 4. The composition ofclaim 1, wherein the neoepitope comprises a polypeptide with a sequenceof amino acids beginning at least the first three amino acids at theamino terminus of SEQ ID NO:
 1. 5. The composition of claim 4, whereinthe neoepitope comprises a polypeptide having an amino acid sequenceidentified by SEQ ID NO:2.
 6. The composition of claim 4, wherein theneoepitope comprises a polypeptide having an amino acid sequenceselected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4. 7.The composition of claim 1, wherein the protein which is cleaved by theprotease is poly(ADP-ribose) polymerase.
 8. The composition of claim 1,wherein the protease is a caspase.
 9. The composition of claim 8,wherein the caspase is selected from the group consisting of caspase 3and caspase
 7. 10. The composition of claim 1, wherein the cell is amammalian cell.
 11. The composition of claim 10, wherein the mammaliancell is a human cell.
 12. The composition of claim 11, wherein the humancell is a leukemia cell.
 13. The composition of claim 12, wherein theleukemia cell is selected from the group consisting of a HL60 cell and aJurkat cell.
 14. A method of producing an antibody, comprising the stepsof: (a) obtaining a polypeptide, said polypeptide comprising an aminoacid sequence corresponding with the amino terminus produced by cleavageof a protein by a protease, said cleavage occurring during apoptosis;and (b) administering the polypeptide to an animal, thereby inducingproduction of an antibody to the polypeptide.
 15. The method of claim14, further comprising the steps of isolating the antibody to thepolypeptide from the animal, and testing the antibody to ensure it isnot immunoreactive with the protein when not cleaved by the protease.16. An antibody immunoreactive with a neoepitope produced in a cellundergoing apoptosis, said neoepitope comprising an amino terminusproduced by cleavage of a protein by a protease, wherein said antibodyis not immunoreactive with the protein when not cleaved by the protease,said antibody produced by a method comprising: (a) obtaining apolypeptide, said polypeptide comprising an amino acid sequencecorresponding with the amino terminus produced by cleavage of a proteinby a protease, said cleavage occurring during apoptosis; and (b)administering said polypeptide to an animal, wherein said administrationcauses production of the antibody.
 17. A composition comprising anantibody specific to the newly formed amino terminus ofpoly(ADP-ribose)polymerase resulting from cleavage of thepoly(ADP-ribose) polymerase by a caspase.
 18. The composition of claim17, wherein the antibody is a monoclonal antibody.
 19. The compositionof claim 17, wherein the antibody is a polyclonal antibody.
 20. Thecomposition of claim 17, wherein the antibody is produced in an animalafter injection of the animal with a polypeptide of about five to aboutten amino acids, comprising at least three amino acids of the newlyformed amino terminus of poly(ADP-ribose) polymerase.
 21. Thecomposition of claim 17, wherein the antibody is produced in an animalhost after injection of the animal with a polypeptide comprising anamino acid sequence beginning from the amino terminus of SEQ ID NO:1.22. The composition of claim 21, wherein the polypeptide is a 7-merhaving the amino acid sequence of SEQ ID NO:2.
 23. The composition ofclaim 21, wherein the polypeptide is identified by the amino acidsequence selected from the group consisting of SEQ ID NO:3 and SEQ IDNO:4.
 24. The composition of claim 17, wherein the protease is acaspase.
 25. The composition of claim 24, wherein the caspase isselected from the group consisting of caspase 3 and caspase
 7. 26. Thecomposition of claim 17, wherein the antibody is preferentiallyimmunoreactive with poly(ADP-ribose)polymerase after cleavage withcaspase, but not before cleavage therewith.